design of a highly thermotolerant, immunogenic sars-cov-2 … · 2020. 8. 15. · 3rd milestone,...

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Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Sameer Kumar Malladi1, Randhir Singh

2, Suman Pandey

2, Savitha Gayathri

2, Kawkab Kanjo

1,

Shahbaz Ahmed1, Mohammad Suhail Khan

1, Parismita Kalita

1, Nidhi Girish

2, Aditya Upadhyaya

2,

Poorvi Reddy2, Ishika Pramanick

1, Munmun Bhasin

1, Shailendra Mani

3, Sankar Bhattacharyya

3,

Jeswin Joseph4, Karthika Thankamani

4, V. Stalin Raj

4, Somnath Dutta

1, Ramandeep Singh

3,

Gautham Nadig2, Raghavan Varadarajan*

1,5

1Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru, India

2Mynvax Private Limited, ES12, Entrepreneurship centre, SID, Indian Institute of Science,

Bengaluru, India

3Translational Health Science and Technology Institute, NCR Biotech Science Cluster,

3rd Milestone, Gurugram-Faridabad Expressway, Faridabad-121001

4Virology Scientific Research (VSR) Laboratory, School of Biology, Indian Institute of Science

Education and Research Thiruvananthapuram (IISER TVM), Kerala, India.

5Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru, India

*: Corresponding author

Raghavan Varadarajan, Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012

(INDIA), Email: [email protected], PHONE: +91-80-22932612, FAX: +91-80-23600535

.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

The copyright holder for this preprintthis version posted August 16, 2020. ; https://doi.org/10.1101/2020.08.15.252437doi: bioRxiv preprint

mailto:[email protected]

https://doi.org/10.1101/2020.08.15.252437

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract:

Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen

state prior to use. This is a major impediment to deployment in resource-poor settings. Several use

viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing

expertise for these novel, nucleic acid based modalities, especially in the developing world.

Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are

primarily directed against the Receptor Binding Domain (RBD) of the viral spike protein. We

describe a monomeric, glycan engineered RBD protein fragment that is expressed at a purified yield

of 200mg/L in unoptimized, mammalian cell culture and in contrast to a stabilized spike ectodomain,

is tolerant of exposure to temperatures as high as 100°C when lyophilized, and upto 70°C in solution.

In prime:boost guinea pig immunizations, when formulated with the MF59 like adjuvant

AddaVax™, the RBD derivative elicited neutralizing antibodies with an endpoint geometric mean

titer of ~415 against replicative virus, comparing favourably with several vaccine formulations

currently in the clinic. These features of high yield, extreme thermotolerance and satisfactory

immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat

COVID-19.



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Introduction

SARS-CoV-2 is the etiological agent of the on-going COVID-19 pandemic 1,2

. As on 15th

August,2020 there are ~20.7 million infections and ~751,000 deaths worldwide 3. The major surface

protein of SARS-CoV-2 is the spike glycoprotein. Like several other viral surface glycoproteins, it is

a homotrimer, with each protomer consisting of two subunits S1 and S2. The S1 subunit consists of

an N-terminal domain (NTD), linker and Receptor binding domain (RBD), and two small

subdomains SD1 and SD2 4–6

(Figure 1A-1D). The RBD domain of the spike glycoprotein binds to

the cell surface receptor ACE2, followed by endocytosis or fusion mediated via the fusion peptide

located on the S2 subunit 7. Most of the neutralizing antibody responses are targeted to the RBD

8–14,

though very recently, neutralizing antibodies against the NTD have also been identified 15

. It is thus

unclear whether the full length spike or the RBD is a better immunogen.

Over 150 vaccine candidates are under development globally 16

. Some vaccine candidates that have

entered rapidly into clinical phase testing include mRNA vaccine candidates by Moderna (mRNA-

1273), BioNTech (BNT162b1)17

, and CureVac (CVnCoV), a Chimpanzee Adenovirus vector

vaccine by University of Oxford and AstraZeneca (ChAdOx1-S) 18

, a non-replicating adenovirus

type-5 (Ad5) vaccine by Cansino (Ad5-nCoV) 19

, a DNA vaccine by Inovio (INO-4800) 20

,

inactivated virus vaccines by Sinovac (PiCoVacc) 21

and Bharat Biotech (COVAXIN), a native like

trimeric subunit spike protein vaccine by Clover Biopharmaceuticals /GSK/Dynavax (SCB-2019),

and a full length recombinant glycoprotein nanoparticle vaccine by Novavax (NVX-CoV2373) 16,22

.

The majority of the above employ full length spike or the corresponding ectodomain as the antigen.

While there is some encouraging pre-clinical and Phase 1 clinical data, there is no precedent for use

of mRNA or viral vectors, which are the farthest along in clinical development, in mass human

vaccinations. In addition, with inactivated or attenuated virus, there are obvious safety issues that

need careful attention. There are few studies that compare the relative immunogenicity of multiple

vaccine candidates expressed in multiple platforms23

. Herein, we report a mammalian cell expressed,



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glycan engineered, RBD based subunit vaccine candidate (mRBD) formulated with an MF59

equivalent adjuvant. In contrast to an equivalent Pichia pastoris expressed RBD protein formulation,

mRBD elicits titers of neutralizing antibodies in guinea pigs well above the levels required for

protection in non-human primate challenge studies. mRBD expresses at eight-fold higher levels and

is substantially more tolerant to thermal stresses than a stabilized spike ectodomain, without

compromising immunogenicity. These data suggests that it is a promising candidate for further

clinical development.

Results

Design of a recombinant RBD subunit vaccine

The RBD of the spike protein is the major target of neutralizing antibodies 8,10–14,24

. SARS-CoV-2 is

79.6% identical to SARS-CoV-1 sequences 25

. The spike protein of SARS-CoV-2 is 80% identical to

its homolog from SARS-CoV-1. The RBD of SARS-CoV-2 shares 74% amino acid sequence

identity with the RBD of SARS-CoV-1. We hypothesized that a receptor binding domain subunit

derivative that lacks flexible termini as well as unpaired cysteines, and retains the ACE2 receptor

binding site, located within the receptor binding motif (RBM, comprising residues 438-505, Figure

1E) as well as the cryptic epitope recognized by the neutralizing antibody CR3022 would be a good

immunogen. We selected the RBD residues based on SWISS Model structure based modelling of

SARS-CoV-2 sequence, prior to availability of any SARS-CoV-2 spike and RBD-ACE2 complex

structures. Two RBD sequences were shortlisted consisting of residues 331-532 and 332-532 with

retention (m331RBD) or deletion (mRBD/pRBD) of the native glycan at N331 for expression in

mammalian and P. pastoris expression systems respectively. The constructs for mammalian

expression are designated as m331RBD and mRBD, and for Pichia expression, pRBD respectively.

In the past few months, several potent neutralizing antibodies directed against the RBD have been

isolated and it currently appears that virtually the entire exposed surface of the RBD is targeted by



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neutralizing antibodies, with the exception of the C-terminal region distal from the RBM. We have

introduced a glycosylation site at N532 in all the above RBD constructs to mask this region of the

surface (Figure 1C, 1D).

RBD (332-532) is more highly expressed and thermotolerant than a stabilized spike

ectodomain

Mammalian cell expressed m331RBD and mRBD were purified by single step Ni-metal affinity

chromatography from transiently transfected Expi293F culture supernatants. The proteins were

confirmed to be predominantly monomeric by SEC (Figure 2A). Proteins from both the constructs

were pure and were expressed at yields of ~32 ± 8.6 mg/L and ~200 ± 10 mg/L for m331RBD and

mRBD respectively. Removal of the N-terminal glycan in m331RBD by introducing the T333H

mutation resulted in substantially increased expression, similar to that of mRBD, confirming that the

presence of the N-terminal glycan is responsible for reduced yield, as has been observed previously

for SARS-CoV-1 RBD26

. The yield for the mRBD was substantially higher compared to a similar

control RBD derivative from SARS-CoV-1 which had a yield of ~30mg/L. All proteins were

monomeric. In SEC, m331RBD which has an additional glycan, elutes before mRBD (Figure 2A).

Given the higher yield of mRBD, most subsequent studies were carried out with this RBD derivative.

nanoDSF thermal melt studies demonstrated that removal of the N-terminal glycan did not affect

protein stability (Figure 2B). mRBD bound ACE2-hFc with a KD of about ~14.2 nM (Figure 2C) and

the neutralizing antibody CR3022 with a KD of 16nM, confirming that the molecule is properly

folded (Figure 2D). mRBD is digested by trypsin with approximate half-lives of 20 and 60 minutes

at 37 and 4°C (Figure 2E) respectively. The digestion kinetics is unaffected by storage for over a

week at 4°C.

A construct with identical amino acid sequence to mRBD (pRBD) was expressed and purified from

P. pastoris strain X-33 from a stably integrated gene cassette at a yield of ~25 mg/L in shake flasks.



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The Pichia protein is more heterogeneous, extensively glycosylated and elutes at higher molecular

weight than mRBD in both SDS-PAGE and SEC (Supplementary Figure 1A, 1F). The thermal

stability of the Pichia purified immunogen pRBD (Tm: 49.2°C) is similar to mammalian cell

expressed versions (Supplementary Figure 1B). The protein bound with comparable affinity to

ACE2-hFc and CR3022 with KD’s of approximately 23 nM and 30 nM respectively, similar but

slightly higher than corresponding values for mRBD (Supplementary Figure 1D, 1E). Pichia

expressed RBD was similarly stable to thermal stress and proteolysis (Supplementary Figure 1C, 1F,

1G). We also attempted to express the protein in E. coli. The protein expressed well but was

targeted to inclusion bodies. Despite multiple attempts employing a variety of refolding strategies,

we were unable to obtain significant quantities of properly refolded protein, competent to bind ACE2

from E. coli.

The spike ectodomain and full length spike formulations are important SARS-CoV-2 vaccine

candidates 5,6,22

and it is therefore important to compare mRBD with these. We purified the Spike-2P

stabilized ectodomain protein from Expi293F cells by single step nickel chelate affinity

chromatography followed by tag removal with a purified yield of ~25 mg/L culture5. The purified

protein was observed to be trimeric on SEC and bound tightly to ACE2-hFc with little dissociation

(Figure 3A, 3B). Negative-stain EM confirmed that Spike-2P purified by us adopts a native like

elongated trimeric structure (Figure 3C) consistent with available structures determined by Cryo-EM

5,6,15. Spike-2P was rapidly digested by trypsin with approximate half lives of 10 and 30 minutes at

37 and 4°C respectively (Figure 3D-E) yielding multiple RBD containing fragments.

Maintaining a proper cold chain during mass vaccination programs can be challenging in low and

middle income countries 27,28

. The aggregation state of mRBD and Spike-2P proteins was unchanged

upon storage at 4°C, freeze thaw and hour-long 37°C incubation (Supplementary Figure 2A, 2B).

mRBD and Spike were also incubated at various temperatures both in PBS buffer for 60 minutes and

for 90 minutes in the lyophilized state. Protein conformational integrity was then assessed in ACE2



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binding experiments using SPR. In PBS, mRBD is 20°C more stable to thermal stress compared to

Spike-2P (Figure 4A, 4B). Remarkably, lyophilized mRBD was stable to exposure of temperatures

as high as 100°C whereas Spike-2P rapidly lost activity at temperatures above 50°C both in solution

and in the lyophilized state (Figure 4C, 4D). In solution, mRBD thermal unfolding was highly

reversible in contrast to Spike-2P as assessed by repetitive equilibrium thermal unfolding (Figure 4

E, 4F).

AddaVax™ adjuvanted RBD elicits neutralizing antibodies in guinea pigs, functionally

blocking the receptor binding motif

Guinea pigs are a widely used, outbred animal model for respiratory infectious diseases and display

disease susceptibility and immune responses that are more similar to humans than the mouse model

29. Guinea pigs were immunized with mammalian cell expressed mRBD protein adjuvanted with

AddaVax™. AddaVax is a squalene-based oil-in-water emulsion that is a mimetic of MF59. MF59

has an extensive safety record and has been used in millions of people in the context of adjuvanted

influenza vaccines 30

. Animals were primed at day 0 and boosted at day 21 with bleeds at day -1

(Pre-Bleed), day 14 and day 35.

The end point ELISA titers to self-antigen ranged from 1:6400 to 1:102400 after the second

immunization in individual animals (Figure 5A). To further confirm and extend these results, the

study was repeated with the inclusion of two additional groups immunized with Pichia expressed

RBD and mammalian cell expressed Spike-2P in addition to the mRBD (Figure 5A, 5B). Results

with the mRBD were consistent in both studies (Figure 5A). Pichia expressed RBD was as

immunogenic as the mRBD in terms of self-titers (Supplementary Figure 3A, 3B) but sera reacted

poorly with mRBD and Spike-2P. Several studies have now shown that ACE2 competition titers and

neutralizing antibody titers are highly correlated 22,31

. Hence serum competition assays were carried

out (Figure 5C). Endpoint neutralization titers with replicative virus were measured using cytopathic



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effect (CPE) as a readout for infection (Figure 5D) and found to range from 160-1280. Surprisingly,

the pRBD sera were non-neutralizing and poorly cross-reactive with mRBD and Spike-2P proteins

(Figure 5A, 5C, 5D), presumably because of hyperglycosylation of the Pichia expressed protein. In

Spike-2P immunized animals titers were more variable than in mRBD immunized animals though

the difference in neutralization titers did not approach statistical significance. A potential advantage

of using the spike as an immunogen is that it contains neutralization epitopes outside the RBD,

including in the NTD 32

. We therefore probed the Spike-2P sera for NTD titers using a mammalian

cell expressed NTD construct. However, all spike sera had NTD endpoint ELISA titers less than 100.

mRBD elicited serum neutralization titers equivalent to those observed in recent mRNA

immunizations in mice and humans by Moderna and better than those observed with several

adenoviral and inactivated viral vaccines (Figure 5E) 18,19,21,33–3839

.

Discussion

The majority of SARS-CoV-2 vaccine candidates currently in clinical testing use either full length

spike or the corresponding ectodomain as antigen, and most involve relatively new nucleic acid or

viral vector modalities that have not been tested in large scale immunizations. There is an obvious

need for highly expressed, stable, low-cost and efficacious subunit vaccine formulations and for side-

by-side comparisons of different candidates. In the present study we characterized the comparative

yield, stability and immunogenicity of mammalian cell and Pichia expressed RBD, as well as

mammalian cell expressed stabilized Spike-2P protein. All three candidates were successfully

expressed, properly folded and immunogenic. The data clearly indicate mammalian cell expressed,

glycan engineered RBD to be the best of the three immunogens, displaying reversible thermal

unfolding and exceptionally high thermal tolerance, a very important attribute for deployment in low

resource settings. The RBD fragment could also be expressed at high yield in the microbial host

Pichia pastoris, and was properly folded, stable and immunogenic. Interestingly, an alhydrogel

adjuvanted formulation of a related SARS-CoV-1 RBD construct was recently shown to be



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immunogenic and protect mice from SARS-CoV-1 challenge 40

. Unfortunately, in the present study

when pRBD was used as an immunogen, the elicited antibodies were poorly reactive with either the

mammalian cell expressed RBD or the corresponding Spike ectodomain. Further, they failed to block

binding of RBD to the ACE2 receptor, suggesting that further alterations to the Pichia expressed

sequence or adjuvant, use of an alternative Pichia strain, or optimization of growth/fermentation

conditions are required before it can be used as an effective immunogen. Recently, various RBD

derived subunit vaccine candidates have been tested for immunogenicity employing varying

fragment lengths, fusion adaptors (Fc, dimers), and adjuvants. No antibody dependent enhancement

of infection, immunopathologies, or Th2 bias have been observed with the SARS-CoV-2 RBD

subunit derivatives examined so far 40–43

. Three independent studies used RBD-Fc fusions with one

study using RBD residues 331-527, another used RBD-Fc from Sino Biologicals (residues not

mentioned) and a third used a full-length S1-Fc fusion (residues 14-685) reporting viral neutralizing

antibody titers of ~100-400, 1280 and NT50 derived from pseudoviral neutralizations of 378

respectively 41,42,44

. One study employed a week long intra-peritoneal immunization regime that is

difficult to implement in large-scale human vaccination programs 44

. The other studies utilizing

RBD-Fc and S1-Fc 41,42

, employed Freunds adjuvant, again not used in human vaccinations. For the

present mRBD formulation both the IC50 values in the ACE2 competition assay and the viral

neutralization titers were about 2% of the corresponding ELISA endpoint titers, suggesting that a

significant fraction of the elicited antibodies are neutralizing. This is in contrast to results with spike

ectodomain immunogens reported in the recent Moderna vaccine trial where neutralization titers

were less than 0.1% of the endpoint ELISA titers 37

. The mRBD glycan engineered monomer also

induced considerably higher neutralization titers compared to another longer RBD monomer that did

not elicit detectable NAbs in most of the immunized animals after two immunizations in a recently

reported mouse immunization study 43

. When a tandem repeat of the latter molecule was made, this

showed a considerable enhancement in immunogenicity with a GMT NT80 neutralization titer of



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~2435, suggesting that a similar oligomerization strategy could be used in the present case as well.

Multiple studies employing a variety of vaccine formulations and modalities have now demonstrated

that SARS-CoV-2 viral neutralization titers in small animals including mice and guinea pigs are

predictive of immunogenicity in macaques and humans (Figure 5E) 17–20,22,23,33–37,40–43,45

. Despite

promising immunogenicity in several cases, all of the above liquid formulations were either

refrigerated or frozen prior to use. In contrast, mRBD can be stored lyophilized without refrigeration

and is thus a promising, thermotolerant vaccine candidate that can be further evaluated in animal

challenge studies and subsequently in human clinical trials.

Materials and Methods

SARS-CoV-2 RBD, NTD, Spike ectodomain and antibody expression constructs

Two fragments of the SARS-CoV-2 Spike protein (S) (accession number YP_009724390.1)

consisting of the receptor binding domain (RBD) residues 331-532 with an N-terminal glycosylation

site and 332-532 with deletion of the N-terminal glycan site deletion were chosen. Residue N532

was engineered to be glycosylated by introducing an NGS motif at the C-termini of the RBD into

both immunogen sequences. The resulting sequences with an HRV-3C precision protease cleavage

site linked to a 10xHistidine tag by GS linker was codon optimized for human cell expression were

expressed under control of the CMV promoter with a tPA signal sequence for efficient secretion. The

clones were named m331RBD (331-532) and mRBD (332-532). Identical RBD amino acid

sequences to those described above, codon optimized for Pichia pastoris expression were cloned into

the AOX1 promoter containing vector pPICZalphaA, containing a MATalpha signal sequence for

efficient secretion. The resulting clones were named p331RBD (expressing RBD 331-532) and

pRBD (expressing RBD 332-532). In the present study, only pRBD was utilized, based on

expression data for the corresponding mammalian and insect cell expression clones. A spike N-

terminal domain construct (NTD) (residues 27-309 with and L296E mutation) under control of the



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CMV promoter with a tPA signal sequence was also designed. A spike construct, encoding a

stabilized ectodomain with two Proline mutations (Spike-2P) optimized for mammalian cell

expression was obtained from the VRC, NIH 5. Genes for the heavy and light chain of the CR3022

antibody were obtained from Genscript (USA) and cloned into the pcDNA3.4 vector

Purification of recombinant proteins expressed in Expi293F cells.

Transfections were performed according to the manufacturer’s guidelines (Gibco, Thermofisher).

Briefly, one day prior to transfection, cells were passaged at a density of 2x106cells/mL. On the day

of transfection, cells were diluted to 3.0x106cells/mL. Desired plasmids (1μg plasmid per 1mL of

Expi293F cells) were complexed with ExpiFectamine293 (2.7μL of ExpiFectamine293 per 1 μg of

plasmid) and transiently transfected into Expi293F cells. Post 16hr, Enhancer 1 and Enhancer 2 were

added according to the manufacturer’s protocol. Five days post transfection, culture supernatant was

collected, proteins were affinity purified by immobilized metal affinity chromatography (IMAC)

using Ni Sepharose 6 Fast flow resin (GE Healthcare). Supernatant was two-fold diluted with 1xPBS

(pH 7.4) bound to a column equilibrated with PBS (pH7.4). A ten-column volume wash of 1xPBS

(pH7.4), supplemented with 25mM imidazole was performed. Finally, the bound protein was eluted

with a gradient of 200-500mM imidazole in PBS (pH 7.4). The eluted fractions were pooled, and

dialysed thrice using a 3-5kDa (MWCO) dialysis membrane (40mm flat width) (Spectrum Labs)

against PBS (pH 7.4). Protein concentration was determined by absorbance (A280) using

NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam

tool (ExPASy).

Purification of recombinant protein expressed in Pichia pastoris.

20µg of pRBD vector was linearized with the PmeI enzyme by incubating at 37oC overnight (NEB,

R0560). Enzyme was inactivated (65°C, 15min) prior to PCR purification of the linearized product

(Qiagen, Germany). 10µg of linearized plasmid was transformed into Pichia pastoris X-33 strain by



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electroporation as per the manufacturers protocol (Thermo Fisher). Transformants were selected on

Zeocin containing YPDS plates at a Zeocin concentration of 2mg/mL (Thermo Fisher Scientific)

after incubation for 3 days at 30oC.

10 colonies from the YPDS plate were picked and screened for expression by inducing with 1%

methanol, fresh methanol was added every 24 hrs. Shake flasks (50mL) containing 8mL BMMY

media (pH 6.0) each were used for growing the cultures for up to 120 hrs maintained at 30oC,

250rpm. The expression levels were monitored by dot blot analysis with anti-His tag antibodies. The

colony showing the highest expression level was then chosen for large scale expression.

Larger scale cultures were performed in shake flasks by maintaining the same volumetric ratio (flask:

media) as the small scale cultures. The expression levels were monitored every 24 hrs using

sandwich-ELISA.

Cultures were harvested by centrifuging at 4000g and subsequent filtering through a 0.45µm filter

(Sartorius). The supernatant was bound to pre-equilibrated Ni Sepharose 6 Fast flow resin (GE

Healthcare). The beads were washed with 1xPBS (pH 7.4), supplemented with 150mM NaCl and

20mM imidazole. Finally, the His tagged pRBD protein was eluted in 1xPBS (pH 7.4) supplemented

with 150mM NaCl and 300mM imidazole. The eluted fractions were checked for purity on a SDS-

PAGE. Following his, appropriate fractions were pooled and dialyzed against 1x PBS (pH7.4) to

remove imidazole.

Purification of recombinant protein from E. coli

The E. coli expression construct, eInCV01R consisted of residues 331-532 of the RBD expressed

under control of the T7 promoter with an N-terminal His tag in the vector pET15b. eInCV01R was

transformed in both E. coli BL21(DE3) (Novagen) and E. coli SHuffle T7 cells (NEB C3029H).

Following cell growth in Terrific Broth and induction with 1 mM IPTG at an OD600 of 1 at either 30



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or 37C, cells were grown for ten hours. Expression was seen in the insoluble and soluble fractions

in these two strains respectively. Following cell lysis of the SHuffle cells, protein was purified using

Ni-NTA chromatography with a yield of about 1 mg/liter. The protein was aggregation prone and

failed to bind ACE2-hFc. In the case of BL21(DE3), following cell lysis, inclusion bodies were

solubilized in buffer containing 7M Guanidine Hydrochloride and 10mM mercaptoethanol. Protein

was purified using Ni-NTA chromatography under denaturing conditions. Protein was diluted into

refolding buffer containing 0.4 M L -Arginine, 100 mM Tris–HCl (pH 8.0), 2.0 mM EDTA (pH 8.0),

5.0 mM L-glutathione reduced, 0.5 mM L-glutathione oxidized, but precipitated. Refolding in the

absence of redox buffer was also unsuccessful.

Tag removal

The His tag was removed by subjecting proteins to digestion with HRV-3C protease (Protein:

HRV-3C = 50:1) in PBS (pH 7.4) buffer and incubating at 4°C, 16 hrs. The untagged protein was

separated from the remaining tag protein and protease by immobilized metal affinity chromatography

(IMAC) using Ni Sepharose 6 Fast flow resin (GE Healthcare). The unbound tag-free protein was

collected and protein concentration was determined by absorbance (A280) using NanoDrop™2000c

with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy).

SDS-PAGE and western blot analysis:

SDS-PAGE was performed to estimate the purity of the recombinant proteins. Protein samples were

denatured by boiling with sample buffer containing SDS, with or without DTT. For western blotting,

following SDS-PAGE, proteins were electrophoretically transferred onto an Immobilon-P membrane

(Millipore). After transfer, the membrane was blocked with 3% non-fat milk. The membrane was

washed with PBST (1xPBS with 0.05% Tween-20) and incubated with antisera raised against mRBD

in guinea pig (1:100). Following this blot was washed and incubated with α-guinea pig ALP



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conjugated antibody (Sigma) at 1:5000. After washing with 1xPBST, blot was developed by

BCIP/NBT Liquid substrate system (Sigma).

Size exclusion chromatography (SEC)

A Superdex-200 10/300GL analytical gel filtration column (GE healthcare) equilibrated in 1xPBS

(pH 7.4) buffer was used SEC profiles were obtained using a Biorad NGC chromatography system.

The Area under the curve (AUC) was calculated using the peak integrate tool in the Evaluation

platform for various peaks from each run

nanoDSF studies

Equilibrium thermal unfolding experiments of m331RBD (- 10xHis tag), mRBD (- 10xHis tag),

pRBD (- 10xHis tag) and Spike-2P was carried out using a nanoDSF (Prometheus NT.48). Two

independent measurements were carried out in duplicate with 10-44μM of protein in the temperature

range of 15-95°C at 40-80% LED power and initial discovery scan counts (350nm) ranging between

5000 and 10000.

SPR-binding of immobilized ACE2-hFc / CR3022 to Spike-2P and RBD derivatives as analytes

ACE2-hFc and CR3022 neutralizing antibody binding studies with the various RBD derivatives

purified from different expression platforms were carried out using the ProteOn XPR36 Protein

Interaction Array V.3.1 from Bio-Rad. Activation of the GLM sensor chip was performed by

reaction with EDC and sulfo-NHS (Sigma). Protein G (Sigma) at 10µg/mL was coupled in the

presence of 10mM sodium acetate buffer pH 4.5 at 30µl/min for 300 seconds in various channels.

The Response Units for coupling Protein G were monitored till ~3500-4000RU was immobilized.

Finally, the excess sulfo-NHS esters were quenched using 1M ethanolamine. Following this,

~1000RU of ACE2-hFc or CR3022 was immobilized on various channels at a flow rate of 5µg/mL



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for 100 seconds leaving one channel blank that acts as the reference channel. mRBD, pRBD and

Spike-2P were passed at a flow rate of 30µL/min for 200 seconds over the chip surface, followed by

a dissociation step of 600 seconds. A lane without any immobilization was used to monitor non-

specific binding. After each kinetic assay, the chip was regenerated in 0.1 M Glycine-HCl (pH 2.7)

(in the case of the ACE2-hFc assay) and 4M MgCl2 (in case of the CR3022 binding assay). The

immobilization cycle was repeated prior to each kinetic binding assay in case of ACE2-hFc. Various

concentrations of the mRBD (- 10xHis tag) (100nM, 50nM, 25nM, 12.5nM, 6.25nM), pRBD (-

10xHis tag) (100nM, 50nM, 25nM) and Spike-2P (-8xHis tag) (146nM, 73nM, 36.5nM, 18.2nM,

9.1nM) in 1x PBST were used for binding studies. The kinetic parameters were obtained by fitting

the data to a simple 1:1 Langmuir interaction model using Proteon Manager.

SPR-binding of immobilized ACE2-hFc to thermal stress subjected mammalian RBD/ Spike-

2P as analytes.

Mammalian RBD/Spike-2P protein at concentration of 0.2 mg/ml in either 1X PBS or as lyophilized

protein was subjected to thermal stress by incubation at the desired temperature in a thermal cycler

for sixty or ninety minutes respectively. Following this, an SPR binding assay as described in above

was performed to assess the binding response using 100nM of the thermally stressed protein Spike-

2P.

Limited Proteolysis

An isothermal limited proteolysis assay was carried out for mRBD, pRBD and Spike-2P using

TPCK-Trypsin at 4°C and 37°C. Substrate proteins were dialyzed in autoclaved water (MQ) and

reconstituted in the digestion buffer (50mM Tris, 1mM CaCl2 (pH 7.5)). ~100µg of each protein was

subjected to proteolysis with 2µg of TPCK-trypsin (TPCK Trypsin : Vaccine candidate =1:50)

incubated at two different temperatures 4°C and 37°C with equal volumes of sample drawn at



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16

various time points 0, 2, 5, 10, 20, 30 and 60 minutes respectively. The reaction was quenched by

SDS-PAGE loading buffer and incubation at 95°C and analysed by SDS-PAGE.

Guinea Pig Immunizations

Groups of four, female, Hartley strain guinea pigs, (6-8 weeks old, approximately weighing 300 g)

were immunized with 20 µg of purified antigen protein diluted in 50 µl PBS, (pH 7.4), and mixed

with 50 µl of AddaVax™ adjuvant (vac-adx-10) (1:1 v/v Antigen : AddaVax™ ratio per

animal/dose) (InvivoGen, USA). Immunizations were given by intramuscular injection on Day 0

(prime) and 21 (boost). Blood was collected and serum isolated on day -2 (pre-bleed), 14 and 35,

following the prime and boost immunization, respectively. All animal studies were approved by the

Institutional Animal Ethics committee (IAEC) No. RR/IAEC/72-2019, Invivo/GP/084.

ELISA- serum binding antibody end point titers

96 well plates were coated with immunized vaccine antigen and incubated for two hours at 25 °C (4

µg/mL, in 1xPBS, 50 µL/well) under constant shaking (300 rpm) on a MixMate thermomixer

(Eppendorf, USA). ACE2-hFc protein coating was used as a control for antigen immobilization.

Following four washes with PBST (200µl/well), wells were blocked with blocking solution (100 µL,

3% skimmed milk in 1xPBST) and incubated for one hour at 25 °C, 300 rpm. Next, antisera (60µL)

starting at 1:100 dilution with four-fold serial dilutions were added, and plates incubated for 1 hour

at 25 °C, 300 rpm. Three washes with 1xPBST were given (200 µL of 1xPBST/well). Following this,

Rabbit ALP enzyme conjugated to anti-Guinea Pig IgG secondary antibody (diluted 1:5000 in

blocking buffer) (50 µL/well) was added and incubated for 1 hour at 25°C, 300 rpm (Sigma-

Aldrich). Subsequently, four washes were given (200 µL of 1xPBST/well). pNPP liquid substrate

(50 µL/well) (pNPP, Sigma-Aldrich) was added and the plate was incubated for 30 minutes at 37 °C,

300 rpm. Finally, the chromogenic signal was measured at 405 nm. The highest serum dilution which



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17

had a signal above the cut off value (0.02 O.D. at 405 nm) was considered as the endpoint titer for

ELISA.

ACE2-hFc competition ELISA

96 well plates were coated with vaccine antigen and incubated overnight at 25 °C (4 µg/mL in 1x

PBS, 50 µl/well) under constant shaking (300 rpm) on a MixMate thermomixer (Eppendorf, USA).

Ovalbumin (4 µg/mL in 1x PBS, 50 µL/well) coating was used as negative control for mRBD

immobilization. Next, four washes with 1xPBST were given (200µl/well) and wells blocked with

blocking solution (100 µL 3% skimmed milk in 1xPBST) for one hour at 25 °C, 300 rpm. Next, anti-

sera (60µL) starting at a dilution of 1:10 in blocking solution, were added to sera competition wells

and blocking solution alone added to the control wells. Samples were incubated for 1 hour at 25°C,

300 rpm and three washes with 1xPBST were given (200 µL of 1xPBST/well). An additional

blocking step was performed for one hour with blocking solution (100µL) incubated at 25°C,

300rpm. Following this, an excess of ACE2-hFc was added (60µL at 20µg/mL) and samples

incubated for one hour at 25°C, 300rpm. Three washes were given (200 µL of PBST/well). Next,

rabbit ALP enzyme conjugated to anti-Human IgG secondary antibody (diluted 1:5000 in blocking

buffer) (50 µl/well) was added and samples incubated for 1 hour at 25°C, 300 rpm (Sigma-Aldrich).

Four washes were given (200 µL of PBST/well). pNPP liquid substrate (50 µL/well) was added and

the plate was incubated for 30 minutes at 37 °C, 300 rpm. Finally, the chromogenic signal was

measured at 405 nm. The percent competition was calculated using the following equation:

– .

Where, Absorbance (Control) is the Absorbance at 405nm of ACE2-hFc protein binding to RBD in

the absence of sera, Absorbance (Sera dilution) is the absorbance from wells where the serum

dilution is incubated with ACE2-hFc protein and mRBD.



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Sandwich ELISA for monitoring RBD expression

4ug/mL ACE2 in 1xPBS pH 7.4 was coated onto ELISA strips (Thermo Fisher) for 1 hr and then

blocked with 3% BSA solution (1x PBS) for 1 hr at RT. Samples were diluted in the blocking

solution and incubated in the wells for 2 hrs at RT. The wells were incubated with anti-His Antibody

(1:10000 dilution) conjugated with Horseradish peroxidase (HRP) enzyme for 1 hr at RT following

which the reaction was visualized by adding 50µL of the chromogenic substrate, TMB (Thermo

Fisher). The reaction was stopped after 20 mins with 50µL of 1M HCl and the absorbance reading at

450nm was obtained from an ELISA Plate reader. Plates were washed with 1xPBS pH 7.4 after each

step.

Negative Staining sample preparation and visualization by Transmission Electron Microscope

For visualization by Transmission Electron Microscope, Spike-2P sample was prepared by

conventional negative staining method. Briefly, Carbon coated Cu grids were glow discharged for 30

seconds and 3.5 μL of sample (0.1mg/mL) was incubated on the grid for 1 minute. The extra sample

and buffer solution was blotted out and negative staining was performed using 1% Uranyl Acetate

solution for 30 seconds. Freshly prepared grids were air-dried for 30 minutes. The negatively stained

sample was visualized at room temperature using a Tecnai T12 electron microscope equipped with a

LaB6 filament operated at 120 kV using a low electron dose. Images were recorded using a side-

mounted Olympus VELITA (2K2K) CCD camera using defocus ranging from -1.3 to -1.5 and a

calibrated pixel size 2.54 Å/pixel at specimen level.

Reference-free 2D classification using single-particle analysis

The evaluation of micrographs was done with EMAN 2.1 46

. Around 2500 particles projections were

picked manually and extracted using e2boxer.py in EMAN2.1 software. 2D particle projections were

binned by 2 using e2proc2d.py. Reference free 2D classification of different projections of particle

were performed using simple_prime2D of SIMPLE 2.1 software 47



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19

CPE based viral Neutralization assay

Guinea pig sera after two immunizations (prime and boost) along with pre-immune (negative

control) samples were heat inactivated prior to the virus neutralization assay by incubating at 56°C

for one hour. SARS-CoV-2 (Isolate: USA-WA1/2020) live virus, 100TCID50 in a volume of 50µL

was premixed with various dilutions of the serum and incubated at 37°C for one hour in a 5% CO2

incubator. Serial dilutions of the incubated premix of virus-serum was added in duplicates into a 96

well plate containing VeroE6 cells (104/well) and cultured for 48/96 hours. After completion of

incubation, the plates were assessed for virus induced cytopathic effect (CPE) and the neutralization

titre was considered as the highest serum dilution at which no CPE was observed under the

microscope.

Production of Pseudotyped SARS-CoV-2 and pseudovirus neutralisation assay

The full-length synthetic construct of Spike glycoprotein of SARS-CoV-2 (GenBank: MN908947)

was synthesized from Genewiz, UK. The complete coding sequences of the spike genes of SARS–

CoV-2 and SARS-CoV (GenBank: AY278491) lacking the endoplasmic retention signal sequence

were amplified from either the synthetic construct or cDNA and cloned into pCAGGS expression

vector (pCAGGS-SARS-2-S and pCAGGS-SARS-S). Pseudotyped coronaviruses were produced as

previously described. Briefly, the plasmids pCAGGS-SARS2-S and pCAGGS-SARS-S were

transiently expressed on HEK 293T cells using Polyethylenimine (PEI) (Polysciences, USA). 24 h

post-transfection, the cells were infected with VSV∆G/GFP virus, incubated for 1 hour, and cells

were washed thrice with 1xPBS and replaced with DMEM medium containing 1% FCS and

antibiotics. Pseudotyped GFP expressing coronaviruses were harvested from the cell supernatant 24

hpi and concentrated using Amicon columns (Merck). Then the viruses were titrated in Vero E6 cells

and stored at -80 °C. A Pseudovirus neutralization assay was performed as described elsewhere with

minor modifications 48,49

. Guinea pig sera obtained after the first immunization were tested at a



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dilution of 1:10-1:80 for the presence of neutralizing antibodies to SARS-CoV-2 using pseudotyped

virus. Briefly, Vero E6 cells (10,000 cells/well) were plated in a 96 well plate (Nunc, Thermo

Scientific) the day before the neutralization assay. Two-fold serially diluted sera were prepared in

96-well plates, starting at 1:10 dilution. Pseudotyped SARS-CoV2 was diluted in Dulbecco's

Modified Dulbecco's Medium (DMEM) supplemented with 1% FBS and penicillin-streptomycin.

Next, 50μl pseudotyped SARS-CoV-2 was added in each well of plates, and the plates were

incubated 37°C for 1h. SARS-CoV pseudotyped virus and SARS-CoV polyclonal antibodies (that

cross-react with SARS-CoV-2) were used as a positive control. Subsequently, serum-pseudovirus

mixtures were transferred to a plate containing Vero E6 cells for one hour. Then the cells were

washed twice with 1xPBS and once with medium, and cells were grown in fresh DMEM medium

followed by incubation in a 5% CO2 environment at 37°C for 24 hours. The neutralization titer was

measured by calculating the percentage of GFP positive cells in each well.

Declaration of Competing Interest

A provisional patent application has been filed for the RBD formulations described in this

manuscript. S.K.M, S.A., R.V, S.P, R.S are inventors. G.N, R.V are founders of Mynvax and S.P,

R.S, N.G, A.U, and PR are employees of Mynvax Private Limited.

Acknowledgements

We thank Dr. Neil King for kindly providing the ACE2-hFc fusion protein and Drs. Lynda Stuart,

Dr. Harry Kleanthous of the Bill and Melinda Gates foundation for helpful discussions. We thank Dr.

Barney Graham for kindly providing the Spike-2P construct. This work was funded by a grant from

the Bill and Melinda Gates Foundation funding to RV. The following reagent NR-52281, SARS-



https://doi.org/10.1101/2020.08.15.252437


21

Related Coronavirus 2, Isolate USA-WA1/2020 was deposited by the Centers for Disease Control

and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2,

Isolate USA-WA1/2020. We also acknowledge funding for infrastructural support from the

following programs of the Government of India: DST-FIST, UGC Center for Advanced Study,

MHRD-FAST, the DBT-IISc Partnership Program, and of a JC Bose Fellowship from DST to RV.

S.K.M acknowledges the support of MHRD-IISc doctoral fellowship. M.B. acknowledges the

support of CSIR doctoral fellowship. N. Sivaji is acknowledged for his enthusiastic technical

support. RS, SM and SB acknowledge the intramural funding received from THSTI under

Translational Research Program grant.

Author Contributions

R.V., S.K.M., conceptualized the work, designed the studies. S.P., R.V., G.N., planned the animal

studies. R.S., P.R., A.U., performed ELISA and ACE2-hFc competition experiments. S.K.M., K.K.,

S.P., N.G., P.K. performed mRBD, Ace2 and Spike protein expression and characterization. S.G.

performed pRBD protein expression and characterization. M.S.K., performed E.coli RBD expression

and purification. S.A. expressed and characterized the mRBD 333H mutant with assistance from

M.S.K. I.P., S.D. provided the EM data and analysis. S.M., S.B., Ram.S. provided CPE neutralizing

antibody assay data. J.J., K.T., V.S.R, performed pseudovirus neutralization assays. M.B. contributed

to antibody epitope analyses. S.K.M wrote the manuscript with contributions from each author.

S.K.M, R.V., G.N. led the studies and edited the paper along with all co-authors.



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Figure 1: S-protein domain organization, structure of Spike and receptor binding domain of

SARS-CoV-2. A) Linear map of the S protein spike with the following domains: NTD, N- terminal

domain; L, linker region; RBD, receptor-binding domain; SD, subdomain; UH, upstream helix; FP,

fusion peptide; CR, connecting region; HR, heptad repeat; CH, central helix; BH, β-hairpin; TM,

transmembrane region/domain; CT, cytoplasmic tail. B) Spike ectodomain trimer highlighting

protomer with RBD in the ‘up’ conformation, NTD in dark blue, RBD in brick red, SD1 and SD2 in

green and S2 subunit in magenta (PDB: 6VSB) C) Epitopes for known RBD directed neutralizing

antibodies. The N and C termini of the receptor- binding motif (RBM) are labeled and in green.

Residues at the binding interfaces with hACE2 are in cyan. The B38 epitope has considerable

overlap with the hAce2 interface, non-overlapping residues are in light blue. Epitopes for S309, P2B-

2F6 are in orange and yellow. Epitope for CR3022 is in pink, this overlaps substantially with the

potent neutralizing antibody H014. The conserved N-glycosylation site at 343 and the engineered,

immune masking glycosylation site at 532 are shown in red. D) Exposed residues with solvent

accessible surface area that are not part of any neutralizing epitope identified so far are shown in

dark blue. The largest such stretch is at the C-terminus where the engineered glycosylation site is

placed. E) Sequence alignment of SARS-CoV-1 (residues: 318-518) and SARS-CoV- 2 (residues

331-532), the blue line indicates the receptor binding motif (RBM), the grey highlight indicate

residues conserved in both SARS-CoV-1 and SARS-CoV-2 and the blue asterisks indicate the ACE2

binding residues. (PDB: 6M0J)

14 685 686 1273816319 541

A

B C

E

SARS-CoV-2 331 NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL 390

SARS-CoV-1 318 NITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDL 377

SARS-CoV-2 391 CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN 450

SARS-CoV-1 378 CFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYN 437

SARS-CoV-2 451 YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV 510

SARS-CoV-1 438 YKYRYLRHGKLRPFERDISNVPFSPDGKPCTP–PALNCYWPLNDYGFYTTTGIGYQPYRV 496

SARS-CoV-2 511 VVLSFELLHAPATVCGPKKSTN 532

SARS-CoV-1 497 VVLSFELLNAPATVCGPKLSTD 518

* ** *

* * * * ** ** ** * ** ***

RBM

RBM

* ** * *

D



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29

Figure 2: Characterization of mammalian cell expressed RBD A) Size exclusion chromatography

profile of m331RBD, mRBD immunogens with predominantly monomeric peak at ~16.0 and

~16.3mL respectively on S200 10/300GL column calibrated with Biorad gel filtration marker

(Cat.No. 1511901) run at flowrate of 0.5mL/min with PBS (pH 7.4) as mobile phase B) nanoDSF

equilibrium thermal unfolding of m331RBD and mRBD. C) SPR binding sensorgrams to ACE2

receptor. The concentrations of mRBD used as analytes are 100nM, 50nM, 25nM, 12.5nM, 6.25nM

D) SPR binding sensorgrams of mRBD with the neutralizing antibody CR3022. mRBD analyte

concentrations are 50nM, 25nM, 12.5nM, 6.2nM, and 3.1nM. E) Limited proteolysis of purified

mRBD protein by TPCK treated trypsin (RBD:TPCK Trypsin=50:1) at 4°C and 37°C.

kon: 6.9x105 M-1s-1

koff: 1.1x10-2 s-1

KD: 15.9x10-9 M

-200 0 200 400 600 800

CR3022

A

C

B

kon: 7.1x105 M-1s-1

koff: 1.0x10-2 s-1

KD: 14.2x10-9 M 200

0

-200 0 200 600 800

100

Res

po

nse

Un

its

(RU

)

Time (s)

ACE2-hFc

D

0 2 5 10 20 30 60 L 0 2 5 10 20 30 60

37 C 4 C

250150100

75

50

37

25

20

15

10

kDadtt + + + + + + + + + + + + + +

E

Temperature (oC)

0 20 40 60 80 100

Fir

st D

eriv

ati

ve

(35

0n

m/3

30

nm

)

0.000

0.005

0.010

0.015

0.020

0.025

0.030m331RBD (Tm: 50.8 ± 1.5 °C)mRBD (Tm : 50.3 ± 0.8 °C)

Elution volume (ml)

0 5 10 15 20 25

UV

28

0 A

bso

rba

nce

(m

AU

)

0

20

40

60

80

100

120

140m331RBD

mRBD

0

100

200

300



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Figure 3: Characterization of mammalian cell expressed Spike-2P A) Size exclusion

chromatography profile of Spike- 2P ectodomain with a trimeric peak at ~8.9mL on S200 10/300GL

column calibrated with Biorad gel filtration marker (Cat. No. 1511901) run at flowrate of

0.75mL/min with PBS(pH 7.4) as mobile phase. B) SPR binding sensorgrams of Expi293F purified

Spike-2P with immobilized ACE2-hFc. The concentrations of Spike-2P analyte used are 146nM,

73nM, 36.5nM, 18nM, 9nM. C) Negative staining EM images of Spike-2P protein. TEM images

indicate that the sample is homogeneous and monodisperse. Representative 2D reference free class

averages of Spike-2P protein. Well-defined class averages indicate that the Spike-2P sample has a

stable and ordered structure and enlarged views of two class averages show the S1 and S2 subunits of

spike protein. D) SDS-PAGE Coomassie stained gel following limited proteolysis of purified Spike-

2P by TPCK treated trypsin (RBD:TPCK Trypsin=50:1) at 4°C and 37°C. E) Western blot following

limited proteolysis of purified Spike-2P by TPCK treated trypsin (RBD:TPCK Trypsin=50:1) at 4°C

and 37 C, probed by α-mRBD guinea pig sera. The red arrow denotes the expected position of the

RBD.

Elution volume (ml)

0 5 10 15 20 25

UV

28

0 A

bso

rba

nce

(m

AU

)

0

20

40

60

80Spike-2P

kon : 2.4x104 M-1s-1

koff : ND*

KD : ND*

Res

po

nse

Un

its

(RU

)

-200 0 200 400 600 8000

200

400

Time (s)

A C

B

D

100 nm

S1

S2

S1S2

25015010075

50

37

25

20

kDa

4 C37 C

0 2 5 10 20 30 60 0 2 5 10 20 30 60

+ + + + + + + L + + + + + + +0 2 5 10 20 30 60 0 2 5 10 20 30 60

+ + + + + + + L + + + + + + +β-ME

4 C37 C

E



https://doi.org/10.1101/2020.08.15.252437


31

Figure 4: RBD and Spike protein functionality upon subjecting to thermal stress. SPR

sensorgrams of ACE2 binding by A), B) protein in 1X PBS, subjected to thermal stress for 60

minutes C), D) Lyophilized protein subjected to thermal stress for 90 minutes. 100nM of Spike-2P

and mRBD were used as analytes. E), F) Equilibrium thermal unfolding measured using nanoDSF

for Spike-2P and mRBD. The initial and repeat unfolding scans are in red (U1) and blue (U2)

respectively.

4 9950 60 70Temperature ( C)

400

200

0-200 0 200 400 600 800

Spike-2P

160

120

80

40

0-200 0 200 400 600 800

mRBD

30 40 50 60 70Temperature ( C)

-200 0 200 400 600 800

200

100

0

300

Spike-2P

-200 0 200 400 600 800

0

40

80

160

mRBD

Res

po

nse

Un

its

(RU

)

Time (s)

Protein in 1X PBS

Lyophilized protein

A

C

B

D

FE

Temperature (oC)

30 40 50 60 70

Fir

st D

eriv

ati

ve

(35

0n

m/3

30

nm

)

0.000

0.005

0.010

0.015

0.020

0.025mRBD U1

mRBD U2

Temperature (oC)

30 40 50 60 70

Fir

st D

eriv

ati

ve

(35

0n

m/3

30

nm

)

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

Spike-2P U1

Spike-2P U2



https://doi.org/10.1101/2020.08.15.252437


32

Figure 5: Comparative immunogenicity data. A-D) Guinea Pig serum titers obtained

after two immunizations with AddaVax™ formulated immunogens. A), B) ELISA endpoint titer

against mammalian cell expressed RBD and Spike ectodomain respectively. C) 50% Inhibitory titers

of ACE2 receptor competing antibodies from animals immunized with mRBD, pRBD and Spike-2P

respectively. Competition titers below 10 are uniformly assigned a value of 5. The dashed line

represents the value 10. D) Endpoint neutralization titers in a cytopathic effect (CPE) assay against

infectious SARS-CoV2, Isolate USA- WA1/2020. The dashed line represents the value 10. ( )Sera

from the first batch of animals immunized sera with mRBD, ( ) second batch of animals

immunized with mRBD, ( ) animals immunized with pRBD, ( ) immunized with mammalian

expressed Spike-2P. E) Live virus neutralization titers for various vaccine candidates in mice,

macaques and humans. mRNA-1273: mRNA vaccine expressing full length Spike-2P protein

assayed by PRNT. BNT162b1: nucleoside modified mRNA vaccine expressing RBD subunit fused to

T4 Fibritin derived Foldon trimerization domain assayed by PRNT. NVX- CoV2373: Full length

Spike-2P adjuvanted protein vaccine assayed by CPE. ChAdOx1 nCoV-19: Replication-deficient

Chimpanzee Adenovirus vector expressing spike protein assayed by PRNT and Marburg VN. Ad5-

ncov: Replication- defective Adenovirus type 5 vector expressing spike protein assayed by CPE.

Ad26.COV2.S: Replication-defective Adenovirus type 26 vector expressing spike protein assayed by

PRNT.

Figure 6

E

1

10

100

1000

10000

100000Mice Macaque Human

100

101

102

103

104

105

Neu

trali

zati

on

Tit

er



https://doi.org/10.1101/2020.08.15.252437


33

Supplementary Figure 1: Characterization of pRBD A) Comparison of SEC profiles of Pichia

and mammalian cell expressed RBD proteins with predominantly monomeric peaks at ~14.5 mL,

~17.0 mL and ~16.3 mL respectively on an S200 10/300GL column calibrated with Biorad gel

filtration marker (Cat. No. 1511901) run at flowrate of 0.5mL/min with PBS (pH 7.4) as mobile

phase B) nanoDSF equilibrium thermal unfolding of pRBD and mRBD. C) Size exclusion

chromatography profile of pRBD following freeze thaw, incubation at 37°C for one hour and stored

overnight at 4°C. D) SPR binding sensorgrams of pRBD to ACE2 receptor. The concentrations of

pRBD used as analytes are 100nM, 50nM, 25nM. E) SPR binding sensorgrams of pRBD with the

neutralizing antibody, CR3022.The concentrations of pRBD used as analytes are 12.5nM, 6.2nM,

3.1nM. F) Coomassie stained Reducing SDS-PAGE of pRBD incubated at various temperatures 4-

4°C stored protein, -80 –80°C frozen and thawed protein, 37- protein incubated at 37°C for 1 hour

without glycerol, 37G- protein incubated at 37°C for 1 hour with 5% glycerol. G) Limited

proteolysis of pRBD with TPCK treated trypsin (RBD:TPCK Trypsin=50:1) at 4°C and 37°C.

D

Elution volume (ml)

0 5 10 15 20 25

UV

28

0 A

bso

rba

nce

(m

AU

)

0

20

40

60

80

100pRBD, 4°C

pRBD, -80°C

pRBD, 37°C

Temperature (oC)

0 20 40 60 80 100

Fir

st D

eriv

ati

ve

(35

0n

m/3

30

nm

)

0.00

0.01

0.02

0.03 pRBD (Tm: 49.2 ± 0.1o

C)

E

kon: 4.1x105 M-1s-1

koff: 9.4x10-3 s-1

KD: 22.9x10-9 M

-200 0 200 400 600 800

0

100

200

ACE2-hFc

Res

po

nse

Un

its

(RU

)

Time (s)

75

2520

15

3750

kDa L 0 2 5 10 20 30 60 L 0 2 5 10 20 30 60

4 C

dtt + + + + + + + + + + + + + +

37 C

B

Elution Volume (ml)

0 5 10 15 20 25

UV

28

0 A

bso

rba

nce

(m

AU

)

0

20

40

60

80

100

120

mRBD

pRBD

A

4 -80 37 37G L kDa

25

3750

75100

20

dtt + + + +

15

10

Incubation temperatureF

CR3022

kon: 9.8x105 M-1s-1

koff: 2.9x10-2 s-1

KD: 29.5x10-9 M

80

40

0

-200 0 200 400 600 800

G

C



https://doi.org/10.1101/2020.08.15.252437


34

Supplementary Figure 2: Additional characterization of mRBD and Spike-2P. Coomassie

stained reducing SDS-PAGE of A) mRBD and B) Spike-2P after incubation under different

conditions: D- Dialysed and stored overnight at 4°C, 4- 4°C stored protein, -80°C frozen and thawed

protein, 37- protein incubated at 37°C for 1 hour without glycerol, 37G- protein incubated at 37°C for

1 hour with 5% glycerol.

D 4 -80 37 37G L

Incubation temperature

dtt + + + + + kDa

25015010075

50

37

25

20

15

A

D

250

150

10075

50

37

4 -80 37 37G L kDa

Incubation temperature

dtt + + + + +

B



https://doi.org/10.1101/2020.08.15.252437


35

Supplementary Figure 3: Guinea Pig Serum titers obtained after two immunizations with

AddaVax™ formulated pRBD. A) ELISA endpoint titer against pRBD B) 50% Inhibitory titers of

ACE2 receptor competing antibodies from guinea pigs immunized with AddaVax™ adjuvanted

pRBD. Competition values below 10 are uniformly assigned a value of five. The dashed line

represents the value 10.

A B



https://doi.org/10.1101/2020.08.15.252437


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